In the fields of medicine and clinical chemistry, many studies and determinations of physiologically reactive species, e.g. cells, proteins, enzymes, cofactors, nucleic acids, substrates, antigens, antibodies, etc. are carried out using "labels" which facilitate the detection or separation of the materials under observation at low concentrations. In one such application, the diagnosis of pathological conditions and the detection of drugs or narcotics in humans and animals is often carried out using labeled materials in specific binding assays using competitive binding principles.
Whenever labels are used, sensitivity is of prime importance due to the generally low levels of biological species that are measured. Procedures carried out using radiometric labels generally do not have sufficient sensitivity for many low level analytes. In addition, radiometric labels suffer from the drawbacks of short useful life and handling hazards.
Labeling with magnetic iron oxide has also been proposed, as described in U.S. Pat. No. 4,452,773 (issued June 5, 1984 to Molday). The sensitivity of such labels is also limited, and their use in labeling biological species requires expensive equipment and tedious procedures.
Fluorescent spectroscopy, one of the most sensitive and versatile of the optical analytical techniques, has become increasingly popular in recent years to overcome the drawbacks of other labeling techniques. In fluorescence spectroscopy, a sample containing a fluorescent species is irradiated with light of known spectral distribution within the excitation spectrum of the target fluorescent species. The intensity of the resulting characteristic emission spectrum of the fluorescent target molecules is determined and is related to the number of target molecules present in the sample. Fluorescent spectroscopy is used extensively for studies of protein structure, bacterial cell wall reactions and conformational changes in enzymes, as well as for determinations of an immunologically reactive ligand in a specific binding assay.
Fluorescent labels comprising chelates of a rare earth element incorporated into polymeric particles of a latex are described in U.S. Pat. Nos. 4,259,313 (issued Mar. 31, 1981 to Frank et al) and related 4,283,382 (issued Aug. 11, 1981 to Frank et al). These labels exhibit improved efficiency in fluorescence and are particularly useful for immunoassays. The polymeric particles serve as carriers for immunologically reactive species directly attached thereto.
Although these labels represent a breakthrough in clinical chemistry because they have improved fluorescence efficiency, there is a need to render them more stable in aqueous solutions. The labels of Frank et al tend to agglutinate spontaneously and to settle out of solution. They therefore have a shortened storage life. They also demonstrate a tendency to agglutinate prematurely during an assay.
Attempts to improve the stability of fluorescent labels led our colleagues, B. A. Burdick and S. J. Danielson to discover improved fluorescent labels and labeled species which are the subject of copending and commonly assigned U.S. patent application Ser. No. 713,206, filed on Mar. 18, 1985 now U.S. Pat. 4,719,182 and entitled FLUORESCENT LABELS AND LABELED SPECIES AND THEIR USE IN ANALYTICAL ELEMENTS AND DETERMINATIONS. The novel materials of that application comprise a rare earth chelate incorporated into a polymeric particle like those of the Frank et al references noted above, but with the critical difference that a polysaccharide arm serves as a linkage between the polymeric particle and the physiologically reactive species. The polysaccharide arm provides improved stability in aqueous solutions which the Frank et al labels generally lack.
Despite the advantages of the materials containing the polysaccharide arm, it is substantially tedious and expensive to make them. Manufacture requires additional starting materials and additional steps of attaching the polysaccharide arm to both the polymeric particle and to the physiologically reactive species. It would be extremely desirable to achieve increased stability of fluorescent labels without the use of the polysaccharide linking arm and the problems associated with it.